EP0866120B1 - Dispositif de culture et méthode d'utilisation - Google Patents

Dispositif de culture et méthode d'utilisation Download PDF

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Publication number
EP0866120B1
EP0866120B1 EP98302072A EP98302072A EP0866120B1 EP 0866120 B1 EP0866120 B1 EP 0866120B1 EP 98302072 A EP98302072 A EP 98302072A EP 98302072 A EP98302072 A EP 98302072A EP 0866120 B1 EP0866120 B1 EP 0866120B1
Authority
EP
European Patent Office
Prior art keywords
plate
cover
lid
orifice
wells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
Application number
EP98302072A
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German (de)
English (en)
Other versions
EP0866120A2 (fr
EP0866120A3 (fr
Inventor
Tadeusz A. Tyndorf
Timothy A. Stevens
Susan L. Barker
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Becton Dickinson and Co
Original Assignee
Becton Dickinson and Co
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
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Publication of EP0866120A2 publication Critical patent/EP0866120A2/fr
Publication of EP0866120A3 publication Critical patent/EP0866120A3/fr
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Publication of EP0866120B1 publication Critical patent/EP0866120B1/fr
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Expired - Lifetime legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/24—Gas permeable parts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/12—Well or multiwell plates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/38—Caps; Covers; Plugs; Pouring means
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/02—Membranes; Filters
    • C12M25/04—Membranes; Filters in combination with well or multiwell plates, i.e. culture inserts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/04—Filters; Permeable or porous membranes or plates, e.g. dialysis

Definitions

  • This invention relates to an apparatus and procedures for growing cells or tissue culture in vitro and more particularly to a culture vessel assembly having means for varying the gas diffusion rate into and out of the assembly and for substantially minimizing biological contamination of the cells or tissue culture in the assembly.
  • Culture vessels are described in U.S. Patent Nos. 4,495,289, 5,026,649, 5,358,871 and 5,366,893 and European Patent Application No. 0 483 620 A2 and U.K. Patent Application No. GB 2 268 187 A.
  • Culture vessels comprise wells which generally have a circular shape and size which permits the introduction therein of a cell culture insert having a membrane upon which cell attachment, growth and differentiation occur.
  • the culture vessels containing the wells are typically rectangular and have a standard size in order to accommodate standard analytical apparatus.
  • Cell culture inserts used in culture vessels are described in U.S. Patent Nos. 4,871,674, 5,026,649 and 5,366,893.
  • the culture of cells is very dependent upon the ability to supply sufficient oxygen to the cells without causing cellular damage.
  • the supply of oxygen for cell respiration is from the atmosphere in the header space above the cells via the liquid culture medium.
  • Aeration of the culture by for example, sparging, surface aeration, medium perfusion, can increase the oxygen availability, however such methods can cause cellular damage.
  • Silicone rubber tubing has been used to improve gas exchange in cell culture, by running the tubing between the well and the atmosphere, but this method is inconvenient and may cause contamination to the cell suspension.
  • WO 95 27196 describes a multi-well plate which prevents cross-contamination of samples through the use of a resilient gasket with covers a majority of the top of the plate and is compressed by a lid having a clamp assembly. It thus provides a sealing assembly for arrays of containers of any size or shape.
  • the gaskets may be unitary sheets with or without an array of openings corresponding to the well opening or may consist of discrete single well gaskets.
  • a multi-tube array is also provided which can be sealed without the need of a gasket or tight-fitting caps.
  • a multi-well plate is also disclosed in which samples can be gas-equilibrated without the risk of microbial contamination.
  • US 1, 515, 016 describes a container where the top of the container is provided with air inlet openings which are protected by a pad of compacted non-absorbent cotton or other suitable fibrous material held against the top of the container in position to cover and protect the openings by means of a plate which in the construction shown is secured to the container wall by means of end lugs or prongs extending through the top of the container with their ends upset on the opposite side to hold the plate securely in place with the cotton pad pressed between the container wall and the plate.
  • the filter pad and the holding plate are on the outside of the container, but they need not necessarily be so placed.
  • the plate need not be perforated provided there is a sufficient open space between its edge and the container wall, and in either case the filer pad is protected against contact with the culture and access of the bacteria to such extent that the filter material, even if of a substance such as cotton which the bacteria will attack, will remain in condition for serving its function for a ver long period of time.
  • WO 89 06272 relates to a container for use in culturing living cells includes an enclosable portion, or base, and a cover for enclosing the enclosable portion, a path defined between the junction of the enclosable portion and the cover and a gas permeable filter positioned within the path. Gases flow into and out of the container through the filter. Vapour phase hydrogen peroxide is degraded into water vapour and oxygen by a material associated with the filter to prevent hydrogen peroxide for entering the container.
  • US 5,602,028 provides method and apparatus for growing cell cultures consisting of a plurality of layers includes a chamber for submerging cells in a medium while supporting and binding them to a semi-permeable membrane.
  • a stirrer is positioned within the chamber and continuously stirs the medium to constantly circulate it past the exposed surface of a semi-permeable membrane on one side of the cell culture growing on the membrane and remote from the membrane to transfer nutrients to both surfaces of the cell either directly or through the membrane without disrupting the cell culture on the membrane.
  • multi-layered cell cultures so grown while still adhered to the membrane and growing is then mounted in a partition separating a pair of chambers to provide substantially the sole passage from one chamber tot he other by flow through the multi-layer culture while in the living state whereby the rate of penetration or degree of penetration of a culture being tested by the material being tested may be measured by measuring the quantity of material in one chamber versus the quantity of that material in the other chamber.
  • an assembly comprising:
  • the present invention further provides a lid for use in conjunction with a lid for use in conjunction with a tissue culture plate comprising:
  • the present invention is an assembly for growing cells or tissue culture in vitro.
  • the assembly comprises a test plate with wells shaped to accommodate an insert, such as a cell culture insert, or for receiving tissue culture media and a removable lid.
  • the test plate preferably comprises an upper surface, a lower surface, and a plurality of wells. Each well is substantially disposed between the upper and lower surface of the test plate.
  • the assembly further includes a removable lid which can be positioned over the upper surface of the test plate.
  • the lid includes a top wall and a peripheral skirt that extends downwardly from the top wall. The lid serves to prevent the loss of sample from the well interior, to protect the contents of the assembly from the environment and to protect the user from the contents of the assembly should it contain a harmful or potentially harmful material.
  • the test plate and related removable lid may be formed in different sizes and geometric configurations so as to be used with different size and geometric configured cell culture inserts.
  • the removable lid may be formed to be positioned over the upper surface of the plate in one orientation so as to reduce cross contamination between the wells in the event the lid is repositioned over the upper surface of the plate.
  • the plate and the removable lid are preferably made of an optically clear plastic to facilitate viewing of the wells and cell culture inserts.
  • the lid comprises means for allowing gas diffusion into and out of the wells of the plate.
  • the means for allowing the gas diffusion into and out of the wells of the plate is at least one port or orifice. Associated with the port is a gas permeable membrane. Most preferably, the gas permeable membrane is liquid impermeable.
  • the means for selectively occluding the membrane is an impermeable film, preferably a thin impermeable film.
  • a thin film may be removably attached to the top of the lid over the port to restrict exposure of the gas permeable membrane to the atmosphere. The thin impermeable film assists is maintaining the biological security of the environment in the wells because the lid does not have to be lifted or removed from the test plate.
  • the means for selectively occluding the membrane is a peel-away gas impermeable label.
  • the label is plastic.
  • Preferred materials of the label include but are not limited to polymeric substrate resins that are impermeable to gases and liquids.
  • the label may be transparent or color coded.
  • the surface of the label may be such that additional information may be hand written on the label.
  • the label allows the user to control the exchange of gas in the: test plate so that the desired growth of the cell culture can be facilitated in the test plate while still providing a closed system that prevents entry of microbial organisms or other contaminants into the wells of the test plate.
  • the label substantially maximizes gas exchange while minimizing the possibility of contamination in the wells of the test plate.
  • a further advantage is that gas exchange takes place exclusively through the membrane without having to partially open the lid and therefore the sterility and leakage into the assembly is substantially minimized.
  • the label does not compromise sterility or invite leakage into the test plate. Other advantages is that the label is reusable and is economically feasible to manufacture.
  • the assembly of the present invention provides the following advantages: (1) a closed vented environment while maintaining sterility and controlling gas exchange; (2) containment of potentially hazardous samples; and (3) minimizing contamination of specimen in the assembly with environmental/exogenous contaminants such as yeast, bacteria and/or virus.
  • the present invention is particularly advantageous when culturing layers of cells, such as in skin cultures where it is desirable to produce layers of keratinocytes for skin grafting in a sterile and gas exchange controlled environment
  • the present invention allows for the controlled passage of oxygen directly to the dividing cells which lie on the membrane surface of the cell insert.
  • the present invention substantially improves gas diffusion into and out of the assembly while maintaining the sterility of the contents of the multiwell plate and allows adequate gaseous exchange to occur between the wells and the external environment. Accordingly, the assembly of the present invention provides a biologically closed environment so as to optimize conditions for cell culturing procedures whereby entry or exit of biological contaminants, gases and/or liquids is substantially minimized.
  • FIG. 1 is a perspective view of the assembly of the present invention.
  • FIG. 2 is a perspective view of the assembly of the present invention with the lid removed.
  • FIG. 3 is a cross-sectional view of the assembly taken along lines 3-3 of FIG. 1.
  • FIG. 4 is an enlarged, partial sectional view of the assembly of FIG. 3.
  • FIG. 5 is a perspective view of an alternate embodiment of the present invention with a label on the lid covering the orifice.
  • FIG. 6 is a perspective view of an alternate embodiment of the present invention with a label covering the entire lid.
  • FIG. 7 is a perspective view of an alternate embodiment of the present invention showing more than one orifice in the lid.
  • An apparatus 10 for growing tissue culture as shown in FIGS. 1-2 includes a culture vessel plate 20 and a lid 50.
  • culture vessel plate 20 includes an upper surface 22 and lower surface 24 .
  • the culture vessel plate further includes a number of wells 26 each comprising a sidewall 28 extending from upper surface 22 of the plate to lower surface 24 of the plate.
  • Each well comprises a top portion 30 and a bottom portion 36 .
  • Top portion 30 comprises an open end 32 , that extends to bottom portion 36 that comprises a closed end 38 .
  • wells 26 in plate 20 There may be any number of wells 26 in plate 20 , although six. twelve. twenty-four, forty-eight and ninety-six well plates are commonly known and available.
  • FIG. 1 a six-well plate is illustrated, merely for exemplary purposes.
  • Most standard multiwell plates have the wells arranged in orthogonal rows and columns so as to be able to clearly identify the individual wells being used, of course, the arrangement of the wells in plate 20 is not an essential limitation of the present invention, since any arrangement of wells is contemplated by the invention.
  • Plate 20 typically is transparent and may be molded, for example, of polyvinylchloride or polystyrene.
  • plate 20 Surrounding the wells and forming the outside border of plate 20 are sidewalls 40 .
  • plate 20 has six (6) sidewalls.
  • Well known tissue culture plates are rectangle or quadrilaterally shaped, although for purposes of the present invention the plate may be fabricated in any practical configuration.
  • Ledge 21 forms a border around the wells on the upper surface of plate 20 and wherein the sidewalls 40 extend to a position raised above the ledge thereby forming a rim surrounding the ledge.
  • Sidewall 40 comprises a two-step section comprising an upper section 42 and a lower section 46 .
  • Lower section 46 is stepped from upper section 42 by an annular shoulder 44 between the upper and lower sections. Therefore, upper section 42 extends from the upper surface 22 to an annular shoulder 44 and lower section 46 extends from annular shoulder 44 to bottom surface 48 of lower surface 24 .
  • Lower section 46 serves as an annular base for plate 20 when the present multiwell tissue culture apparatus is being used.
  • Lid 50 is a separate, removable member which covers all of the wells of the plate. Although lid 50 is illustrated in all of the figures, the details of lid 50 are best illustrated in FIGS. 2-3.
  • Lid 50 includes a substantially planar cover 52 large enough to extend over all of the wells of the plate planar cover 52 has a top surface 54 and a bottom surface 56.
  • the shape of cover 52 is compatible with the shape of plate 20 .
  • lid 50 is formed to be positioned over the upper surface of the plate in one orientation so as to reduce cross contamination between the wells in the event the user tries to reposition the lid over the plate.
  • skirt 58 Extending around cover 52 is a downwardly depending skirt 58 which forms a border around lid 50 .
  • Skirt 58 has an inner wall surface 60 and an outer wall surface 62 , and six sides each of which extends downwardly from the cover to a bottom stop surface 64 .
  • rib 66 Inwardly, spaced from skirt 58 and extending downwardly from bottom surface 56 of the planar cover is rib 66 . This rib, lying substantially parallel to each of the sides of skirt 58 is positioned on the cover so as to depend downwardly into recessed ledge 21 in the plate.
  • Gas permeable membrane materials may be made from any suitable gas permeable material so long as it provides free passage of gases such as oxygen and carbon dioxide into the test plate while preventing microorganisms such as bacteria and fungi from passing there through.
  • skirt 58 extends towards annular shoulder 44 of the plate thereby leaving a space 68 therebetween annular shoulder 44 and bottom stop surface 64 .
  • FIG. 5 An additional embodiment of the invention as shown in FIG. 5 includes many components which are substantially identical to the components of FIG. 1. Accordingly, similar components performing similar functions will be numbered identically to those components of FIG. 1 except that a suffix "a" will be used to identify those similar components in FIG. 5.
  • a peel-away thin film label 76 is removably attached to cover 52a to selectively occlude membrane 74a of orifice 72a .
  • the film may be fully or partially removed or left in place according to the particular culture requirements.
  • the thin film label material may be made of polyethylene or polyethylene terephthalate and adhesive materials may include acrylic adhesives.
  • optional peel-away thin film label 76 is circular and has a tab 77 for ease of use. Although it is within the purview of the invention to provide a peel-away thin film that is circular, films of different geometries and dimensions may be provided.
  • FIG. 6 An additional embodiment of the invention as shown in FIG. 6 includes many components which are substantially identical to the components of FIG. 2. Accordingly, similar components performing similar functions will be numbered identically to those components of FIG. 2, except that a suffix "b" will be used to identify those similar components in FIG. 6.
  • Label 79 as shown in FIG. 6 is an alternate embodiment of the label that can be used with assembly 10b .
  • the alternate embodiment of the invention as shown in FIG. 6, is that label 79 covers the entire planar cover surface of lid 50b .
  • FIG. 7 An additional embodiment of the invention as shown in FIG. 7 includes many components which are substantially identical to the components of FIG. 2. Accordingly, similar components performing similar functions will be numbered identically to those components of FIG. 2, except that a suffix "c" will be used to identify those similar components in FIG. 7.
  • Lid 50c as shown in FIG. 7 is an alternate embodiment of the lid that can be used with assembly 10c .
  • the alternate embodiment of the invention as shown in FIG. 7, is that two orifices 90 are located in the planar cover and each has a liquid impermeable membrane 92 associated therewith.
  • FIG. 7 only show two orifices, it is well within the purview of the invention to have more than two orifices in the planar cover.
  • the present invention may be used for culturing cells as follows:
  • the culture vessel assembly of the present invention may be comprised of simple moldable parts which may be mass produced from a variety of materials, including, for example, polyethylene, polystyrene, polyethylene terephthalate and polypropylene.
  • materials should be selected which provide a small degree of resiliency for the purpose of providing ease of use for subsequent examination of the developed cultured cells.

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  • Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Sustainable Development (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Clinical Laboratory Science (AREA)
  • Immunology (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Claims (3)

  1. Assemblage comprenant :
    une plaque de test (20) comprenant plusieurs cupules (26) comportant des ouvertures (30) destinées à recevoir un milieu de culture tissulaire et une paroi latérale verticale (28) formant une limite externe de ladite plaque (20);
    un couvercle (50) positionné de façon amovible sur ladite plaque, comprenant une couverture pratiquement plane (52) s'étendant au-dessus desdites cupules (26), une bordure (58) entourant ladite couverture (65) définissant plusieurs coins sur ladite couverture (52) et s'étendant vers le bas, de manière espacée par rapport à ladite paroi latérale (28) de ladite plaque (20) ;
    un orifice (72) dans ladite couverture plane (52) ; et
    une membrane perméable aux gaz (74) associée audit orifice (72) ;
       caractérisé en ce que :
    un film imperméable aux gaz (76) est fixé de manière amovible sur ladite couverture (52) au-dessus dudit orifice (72) et de ladite membrane perméable aux gaz (74), ledit film (76) pouvant ainsi être enlevé complètement ou partiellement pour faciliter la diffusion de gaz dans ledit assemblage (10) et hors de celui-ci ; et
    ladite plaque de test (20) comprend en outre une moulure (21) espacée vers l'intérieur de ladite paroi latérale (28) de ladite plaque (20) et s'étendant autour de ladite plaque (20) entre ladite paroi latérale (28) et lesdites cupules (26), ladite paroi latérale (28) s'étendant vers une position surélevée au-dessus de ladite moulure (21), formant ainsi un rebord entourant ladite moulure (21).
  2. Couvercle (50) destiné à être utilisé en combinaison avec une plaque de culture tissulaire (20), comprenant :
    une couverture pratiquement plane (52) comprenant une surface supérieure (54) et une surface inférieure (56) ;
    une bordure (58) entourant ladite couverture (52) définissant plusieurs coins sur ladite couverture (52) et s'étendant vers le bas pour former des parois latérales ;
    un orifice (72) dans ladite couverture comprenant une membrane perméable aux gaz (74) ;
    un film imperméable aux gaz (76) est fixé de manière amovible sur ladite couverture (52) au-dessus dudit orifice (72) et de ladite membrane perméable aux gaz (74), ledit film (76) pouvant ainsi être enlevé complètement ou partiellement ; et
    plusieurs brides espacées vers l'intérieur de ladite bordure (58) et s'étendant vers le bas à partir de ladite surface inférieure (56) de ladite couverture (52).
  3. Procédé de culture de cellules, comprenant les étapes ci-dessous :
    (a) fourniture d'un assemblage selon la revendication 1 comme récipient de culture ;
    (b) enlèvement dudit couvercle (50) dudit récipient de culture ;
    (c) placement d'un insert cellulaire comprenant une membrane dans ladite cupule (26) dudit récipient de culture ;
    (d) dépôt d'une couche de cellules sur chaque membrane dudit insert cellulaire dans ledit récipient de culture ;
    (e) superposition d'un milieu de croissance à ladite couche de cellules ;
    (f) agencement dudit couvercle (50) sur ladite plaque (20) pour recouvrir lesdites cupules (26) ; et
    (g) ventilation de ladite membrane en assurant des poussées d'accès d'air à travers ledit orifice dans ledit couvercle (50) dudit récipient de culture (10) par enlèvement et remise en place de ladite étiquette (76) au-dessus dudit orifice.
EP98302072A 1997-03-19 1998-03-19 Dispositif de culture et méthode d'utilisation Expired - Lifetime EP0866120B1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US818649 1997-03-19
US08/818,649 US5882922A (en) 1997-03-19 1997-03-19 Culture vessel assembly

Publications (3)

Publication Number Publication Date
EP0866120A2 EP0866120A2 (fr) 1998-09-23
EP0866120A3 EP0866120A3 (fr) 1999-06-30
EP0866120B1 true EP0866120B1 (fr) 2002-12-04

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US (1) US5882922A (fr)
EP (1) EP0866120B1 (fr)
JP (1) JP4108816B2 (fr)
DE (1) DE69809824T2 (fr)

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JPH10262649A (ja) 1998-10-06
EP0866120A2 (fr) 1998-09-23
JP4108816B2 (ja) 2008-06-25
DE69809824T2 (de) 2003-09-11
US5882922A (en) 1999-03-16
DE69809824D1 (de) 2003-01-16
EP0866120A3 (fr) 1999-06-30

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